The side population phenomenon enriches for designated adrenocortical progenitor cells in mice

Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01–0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified.In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished inPbx1haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficientTpit−/−mice was significantly wider compared with wild-type adrenals (Tpit−/−259±10.7 vsTpit+/−100±12.3%;P<0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit−/−0.45±0.16 vsTpit+/−0.13±0.04%;P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%;P=0.33 and 0.09%), providing indirect evidence for a stem cell ‘arrest’ inTpit−/−mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.

Download Full-text

Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin

Biochemical Journal ◽

10.1042/bj2930181 ◽

1993 ◽

Vol 293 (1) ◽

pp. 181-185 ◽

Cited By ~ 40

Author(s):

N J Watkins ◽

A K Campbell

Keyword(s):

Light Emission ◽

Specific Activity ◽

In Vitro Transcription ◽

Wild Type ◽

Proline Residue ◽

Long Term Stability ◽

Recombinant Aequorin

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

Download Full-text

Amino Acid Transporter X Is Required for Hematopoietic Stem Cell Maintenance through Regulating Specific Amino Acids Level

Blood ◽

10.1182/blood.v126.23.1166.1166 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1166-1166 ◽

Cited By ~ 1

Author(s):

Zhenrui Li ◽

Keiyo Takubo ◽

Pengxu Qian ◽

Toshio Suda ◽

Linheng Li

Keyword(s):

Amino Acids ◽

Stem Cell ◽

Amino Acid ◽

Acid Metabolism ◽

Hematopoietic Stem ◽

Cell Pool ◽

Stem Cell Pool

Abstract Hematopoietic stem cells (HSCs) maintenance is required to preserve stem cell pool and compensate the dynamic loss of blood cells. Previous studies of HSCs maintenance mainly focus on the quiescent versus active state of HSCs and accumulated evidence indicates that metabolism plays a critical role in coordinating divergent stem cell states. While recent reports largely emphasized the role of catabolic glycolysis on long-term (LT) HSC maintenance, we found that free amino acids are enriched in primitive stem cell by ~1.5 fold. Given that amino acid metabolism in HSCs is largely unknown, we first cultured bone marrow (BM) cells with individual amino acid deprived medium to study the function of individual amino acids on HSCs in vitro. Surprisingly, we found that specific amino acids, including valine, methionine and threonine (VMT), are essential for maintaining primitive HSCs, as removing them (VMT) individually from media dramatically reduced primitive HSC number by over 95%. Thus, we hypothesize that specific amino acids are critical for preserving the stem cell pool and maintaining their function. To test it, we transplanted equal number of cells cultured with complete or individual VMT deprived media into lethally irradiated recipient mice and found VMT deprivation in vitro impaired stem cell repopulation ability. We also identified the amino acid transporter X (AATX) that is specifically expressed in HSCs and maintain VMT levels within the cell. Furthermore, inhibition of AATX reduced LT-HSC (LSK CD34- Flk2-) number in vivo. BM transplantation indicated that AATX inhibition impaired stem cell long-term reconstitution ability by over 2 fold. Our studies uncovered a role of amino acid metabolism in HSC maintenance and discovered the underlying molecular mechanism related to the amino acid transport. This finding may impact clinical treatment of blood disorders including leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The Role of Apoptosis in the Regulation of Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.191.2.253 ◽

2000 ◽

Vol 191 (2) ◽

pp. 253-264 ◽

Cited By ~ 226

Author(s):

Jos Domen ◽

Samuel H. Cheshier ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Cells ◽

Wild Type ◽

Direct Role ◽

Hematopoietic Stem

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2–transgenic mice have increased numbers of HSC in bone marrow (2.4× wild type), but fewer of these cells are in the S/G2/M phases of the cell cycle (0.6× wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

Download Full-text

Pituitary tumors contain a side population with tumor stem cell-associated characteristics

Endocrine Related Cancer ◽

10.1530/erc-14-0546 ◽

2015 ◽

Vol 22 (4) ◽

pp. 481-504 ◽

Cited By ~ 38

Author(s):

Freya Mertens ◽

Lies Gremeaux ◽

Jianghai Chen ◽

Qiuli Fu ◽

Christophe Willems ◽

...

Keyword(s):

Stem Cell ◽

Cell Line ◽

Pituitary Adenomas ◽

Pituitary Tumor ◽

Side Population ◽

Pituitary Tumors ◽

Stem Cell Marker ◽

Tumor Stem Cell ◽

Tumor Pathogenesis

Pituitary adenomas cause significant endocrine and mass-related morbidity. Little is known about the mechanisms that underlie pituitary tumor pathogenesis. In the present study, we searched for a side population (SP) in pituitary tumors representing cells with high efflux capacity and potentially enriched for tumor stem cells (TSCs). Human pituitary adenomas contain a SP irrespective of hormonal phenotype. This adenoma SP, as well as the purified SP (pSP) that is depleted from endothelial and immune cells, is enriched for cells that express ‘tumor stemness’ markers and signaling pathways, including epithelial–mesenchymal transition (EMT)-linked factors. Pituitary adenomas were found to contain self-renewing sphere-forming cells, considered to be a property of TSCs. These sphere-initiating cells were recovered in the pSP. Because benign pituitary adenomas do not grow in vitro and have failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was further used. We identified a SP in this cell line and found it to be more tumorigenic than the non-SP ‘main population’. Of the two EMT regulatory pathways tested, the inhibition of chemokine (C-X-C motif) receptor 4 (CXCR4) signaling reduced EMT-associated cell motility in vitro as well as xenograft tumor growth, whereas the activation of TGFβ had no effect. The human adenoma pSP also showed upregulated expression of the pituitary stem cell marker SOX2. Pituitaries from dopamine receptor D2 knockout (Drd2−/−) mice that bear prolactinomas contain more pSP, Sox2+, and colony-forming cells than WT glands. In conclusion, we detected a SP in pituitary tumors and identified TSC-associated characteristics. The present study adds new elements to the unraveling of pituitary tumor pathogenesis and may lead to the identification of new therapeutic targets.

Download Full-text

Role of Cell Adhesion in the Maintenance of Hematopoietic Stem Cells in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v104.11.669.669 ◽

2004 ◽

Vol 104 (11) ◽

pp. 669-669

Author(s):

Fumio Arai ◽

Atsushi Hirao ◽

Kentaroh Hosokawa ◽

Toshio Suda

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Adhesion ◽

Side Population ◽

Bone Marrow Niche ◽

Bone Surface ◽

Hematopoietic Stem ◽

Bm Niche

Abstract Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis. We have demonstrated that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and anti-apoptotic, transplantable and comprise a side-population (SP) of HSCs, which contact closely to angiopoietin-1, a ligand for Tie2, expressing osteoblasts (OBs) in the BM niche. Tie2 and Ang-1 are part of a key signaling interaction between HSCs and niche cells. This signaling pathway regulates the feature of HSCs in the BM niche. The interaction of Tie2 with Ang-1 in vitro is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhanced the ability of HSCs to become quiescent and also induced their adhesion to bone surface, resulting protection of HSC compartment from stresses which suppress hematopoiesis (Arai et al., Cell, 2004). In this study, we focused on the role of cell adhesion molecule on the maintenance of HSCs in BM niche. A previous paper reported that both quiescent HSCs and some of osteoblasts express N-cadherin. We found that Tie2+ HSC expressed N- and VE-cadherin, and OBs expressed N-, P-, and OB-cadherin. This suggests that an adherens junction between HSCs and OBs created via N-cadherin may contribute to HSC maintenance. Furthermore, N-cadherin overexpressing OP9 stromal cells (OP9/N-cadherin) maintained LTC-IC more than control OP9 cells did. Overexpression of N-cadherin in HSCs also maintained colony formation. In addition, in the presence of Ang-1 in coculture of OP9/N-cadherin and HSCs facilitated the maintenance of CFU-C and HPP-CFC formation after long-term culture. Furthermore, Ang-1 treatment upregulated the expression of N-cadherin in Tie2+ HSCs as well as β1-integrin. It suggests that Tie2/Ang-1 signaling enhanced cell-cell adhesion between HSCs and OBs. Altogether, these observations led us to the novel model that the localization of HSCs on bone surface is regulated by stem cell specific adhesion molecules such as N-cadherin. Once the HSCs adjacently localize to OBs, Ang-1 produced by OBs may activate Tie2 on the HSCs and promote tight adhesion of HSCs in the niche, resulting in quiescence and enhanced survival of HSCs.

Download Full-text

The stem cell revolution: on the role of CD164 as a human stem cell marker

npj Regenerative Medicine ◽

10.1038/s41536-021-00143-1 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Suzanne M. Watt ◽

Hans-Jörg Bühring ◽

Paul J. Simmons ◽

Andrew W. C. Zannettino

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Binding Sites ◽

Cell Function ◽

Stem Cell Marker ◽

Human Stem Cells ◽

Life Threatening

AbstractAccurately defining hierarchical relationships between human stem cells and their progeny, and using this knowledge for new cellular therapies, will undoubtedly lead to further successful treatments for life threatening and chronic diseases, which represent substantial burdens on patient quality of life and to healthcare systems globally. Clinical translation relies in part on appropriate biomarker, in vitro manipulation and transplantation strategies. CD164 has recently been cited as an important biomarker for enriching both human haematopoietic and skeletal stem cells, yet a thorough description of extant human CD164 monoclonal antibody (Mab) characteristics, which are critical for identifying and purifying these stem cells, was not discussed in these articles. Here, we highlight earlier but crucial research describing these relevant characteristics, including the differing human CD164 Mab avidities and their binding sites on the human CD164 sialomucin, which importantly may affect subsequent stem cell function and fate.

Download Full-text

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2018.3.6 ◽

2018 ◽

Vol 8 (3) ◽

pp. 36-41

Author(s):

Diep Do Thi Hong ◽

Duong Le Phuoc ◽

Hoai Nguyen Thi ◽

Serra Pier Andrea ◽

Rocchitta Gaia

Keyword(s):

First Generation ◽

Good Reproducibility ◽

Complex Matrices ◽

Long Term Stability ◽

In Vitro Characterization ◽

Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

Download Full-text

Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

Applied Sciences ◽

10.3390/app11156865 ◽

2021 ◽

Vol 11 (15) ◽

pp. 6865

Author(s):

Eun Seon Lee ◽

Joung Hun Park ◽

Seong Dong Wi ◽

Ho Byoung Chae ◽

Seol Ki Paeng ◽

...

Keyword(s):

Cold Stress ◽

Wild Type ◽

Rna Chaperone ◽

E Coli ◽

Cold Sensitive ◽

Thioredoxin H ◽

Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

Download Full-text

Suppressive Role of Bam32/DAPP1 in Chemokine-Induced Neutrophil Recruitment

International Journal of Molecular Sciences ◽

10.3390/ijms22041825 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1825

Author(s):

Li Hao ◽

Aaron J. Marshall ◽

Lixin Liu

Keyword(s):

Small Gtpases ◽

Neutrophil Recruitment ◽

Neutrophil Chemotaxis ◽

Wild Type ◽

Adaptor Molecule ◽

Geranylgeranyltransferase I ◽

And Migration ◽

Rap1 Activation

Bam32 (B cell adaptor molecule of 32 kDa) functions in the immune responses of various leukocytes. However, the role of neutrophil Bam32 in inflammation is entirely unknown. Here, we determined the role of Bam32 in chemokine CXCL2-induced neutrophil chemotaxis in three mouse models of neutrophil recruitment. By using intravital microscopy in the mouse cremaster muscle, we found that transmigrated neutrophil number, neutrophil chemotaxis velocity, and total neutrophil chemotaxis distance were increased in Bam32−/− mice when compared with wild-type (WT) mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32−/− mice at 2 but not 4 h. The CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32−/− neutrophils in vitro were increased. We examined the activation of small GTPases Rac1, Rac2, and Rap1; the levels of phospho-Akt2 and total Akt2; and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 suppressed Rap1 activation without changing the activation of Rac1 and Rac2. The pharmacological inhibition of Rap1 by geranylgeranyltransferase I inhibitor (GGTI298) increased WT neutrophil chemotaxis. In addition, the deficiency of Bam32, as well as the inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. The inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32−/− mice. Together, our results reveal that Bam32 has a suppressive role in chemokine-induced neutrophil chemotaxis by regulating Rap1 activation and that this role of Bam32 in chemokine-induced neutrophil recruitment relies on the activation of PI3K effector Akt.

Download Full-text

SiRNA Directed Against Annexin II Receptor Inhibits Angiogenesis via Suppressing MMP2 and MMP9 Expression

Cellular Physiology and Biochemistry ◽

10.1159/000369745 ◽

2015 ◽

Vol 35 (3) ◽

pp. 875-884 ◽

Cited By ~ 16

Author(s):

Hongyuan Song ◽

Dongyan Pan ◽

Weifeng Sun ◽

Cao Gu ◽

Yuelu Zhang ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Umbilical Vein ◽

Tube Formation ◽

Annexin Ii ◽

Mmp9 Expression ◽

Cell Arrest ◽

Umbilical Vein Endothelial Cells

Background/Aims: Annexin II receptor (AXIIR) is able to mediate Annexin II signal and induce apoptosis, but its role in angiogenesis remains unclear. This study tries to investigate the role of AXIIR in angiogenesis and the plausible molecular mechanism. Methods/Results: RNA interference technology was used to silence AXIIR, and the subsequent effects in vitro and in vivo were evaluated thereafter. Our data indicated that human umbilical vein endothelial cells (HUVECs) expressed AXIIR and knockdown of AXIIR significantly inhibited HUVECs proliferation, adhesion, migration, and tube formation in vitro and suppressed angiogenesis in vivo. Furthermore, AXIIR siRNA induced cell arrest in the S/G2 phase while had no effect on cell apoptosis. We found that these subsequent effects might be via suppressing the expression of matrix metalloproteinase 2and matrix metalloproteinase 9. Conclusion: AXIIR participates in angiogenesis, and may be a potential therapeutic target for angiogenesis related diseases.

Download Full-text